The field of the invention is fluorescence spectroscopy and imaging, and in particular, the use of fluorescence to detect epithelial pre-cancers and cancers.
Fluorescence spectroscopy and imaging in the ultraviolet-visible (UV-VIS) wavelength spectrum is an exciting new modality for detecting human epithelial pre-cancer and cancer. Fluorescence spectroscopy is performed by irradiating the tissue surface with light and detecting the fluorescent light emitted by “fluorophores” in the tissue. The fluorophores may be “endogenous” molecules that absorb the impinging photons and emit photons at a different wavelength, or they may be “exogenous” fluorophores such as injected photosensitizing agents. This emerging technology has shown promising results for detecting early neoplastic growth in a variety of organ sites including the colon, bronchus, cervix, oral cavity, skin and bladder. Noninvasive and fast detection of epithelial pre-cancers and early cancers through the use of fluorescence spectroscopy can significantly improve the efficacy and reduce cost of cancer screening and diagnostic programs.
One of the most widely explored applications of fluorescence spectroscopy is the detection of endoscopically invisible, early neoplastic growth in epithelial tissue sites. Early neoplastic growth refers to pre-malignant changes such as dysplasia and carcinoma in situ (CIS), which precede malignancy, i.e., invasive carcinoma. Currently, there are no effective and commonly accepted diagnostic techniques for these early tissue transformations. Fluorescence spectroscopy is ideally suited for this application because of its ability to examine tissue surfaces, rather than tissue volumes, and the ability to deploy this technology in an endoscopic device. If fluorescence spectroscopy can be applied successfully as a diagnostic technique in this clinical context, it may increase the potential for curative treatment, and thus, reduce complications. In addition to the potential for improved patient outcome, the fast and noninvasive nature of this diagnostic technique may also reduce health care costs.
Referring to FIG. 1, when a biologic molecule is illuminated at an excitation wavelength, which lies within the absorption spectrum of that molecule, it will absorb photons' energy and be activated from its ground state (state of lowest energy; S0) to an excited state (state of higher energy; S1). The molecule can then relax back from the excited state to the ground state by generating energy in the form of fluorescence, at emission wavelengths, which are longer than the excitation wavelength. The phenomenon of fluorescence displays several general characteristics for a particular biologic molecule. First, due to losses in energy between absorption and emission, fluorescence occurs at emission wavelengths, which are always red-shifted, relative to the excitation wavelength. Second, the emission wavelengths of fluorescence are independent of the excitation wavelength. Third, the fluorescence spectrum of a biologic molecule is generally a mirror image of its absorption spectrum. The fluorescence of a biologic molecule is characterized by its quantum yield and its lifetime. The quantum yield is simply the ratio of the energy converted to fluorescence to the energy absorbed. The lifetime is defined as the average time the biologic molecule spends in the excited state before returning to the ground state. The fluorescence quantum yield and lifetime are modified by a number of factors that can increase or decrease the energy losses. For example, a molecule may be non-fluorescent as a result of a large rate of non-radiative decay (thermal generation).
Fluorescence spectroscopy is the measurement and analysis of various features that are related to the fluorescence quantum yield and/or lifetime of a biologic molecule (s). The fluorescence intensity of a biologic molecule is a function of its concentration, its extinction coefficient (absorbing power) at the excitation wavelength, and its quantum yield at the emission wavelength. A fluorescence emission spectrum represents the fluorescence intensity measured over a range of emission wavelengths, at a fixed excitation wavelength. Conversely, a fluorescence excitation spectrum is a plot of the fluorescence intensity at a particular emission wavelength, for a range of excitation wavelengths. A fluorescence, excitation-emission matrix (EEM) is a two dimensional contour plot, which displays the fluorescence intensities as a function of a range of excitation and emission wavelengths. Each contour represents points of equal fluorescence intensity. Finally, fluorescence lifetime measurements are represented as the fluorescence intensity distributed over a very short time scale, at a fixed excitation-emission wavelength pair. FIGS. 2a–2d are graphic illustrations of a fluorescence (a) emission spectrum, (b) excitation spectrum, (c) EEM and (d) decay profile.
Table 1 shows a list of biologic endogenous fluorophores and their excitation and emission maxima. These endogenous fluorophores include the amino acids, structural proteins, enzymes and coenzymes, vitamins, lipids and porphyrins. Their excitation maxima range lies between 250 and 450 nm (which spans the ultraviolet and visible spectral range), whereas their emission maxima range lies between 280 and 700 nm (which spans the ultraviolet, visible and near-infrared spectral range). Fluorophores that are believed to play a role in transformations that occur in the neoplastic process in tissue, are the amino acids, tryptophan, the structural protein, collagen, the co-enzymes, NADH and FAD and porphyrins.
TABLE 1EndogenousExcitation MaximaEmission MaximaFluorophores(nm)(nm)Amino acidsTryptophan280350Tyrosine275300Phenylalanine260280Structural ProteinsCollagen325, 360400, 405Elastin290, 325340, 400Enzymes and CoenzymesFAD, Flavins450535NADH290, 351440, 460NADPH336464[NADH, reduced nicotinamide dinucelotide; NAD(P)H, reducednicotinamide dinucleotide phosphate; FAD, flavin adenine dinucleotide.]VitaminsVitamins A327510Vitamins K335480Vitamins D390480Vitamins B6 compoundsPyridoxine332, 340400Pyridoxamine335400Pyridoxal330385Pyridoxic acid315425Pyridoxal5′–330400phosphateVitamin B12275305LipidsPhospholipids436540, 560Lipofuscin340–395540, 430–460Ceroid340–395430–460, 540Porphyrins400–450630, 690
Fluorescence spectroscopy of turbid media such as tissue depends on any of a number of factors. It depends on the concentration and distribution of fluorophore(s) present in the tissue, as well as the biochemical/biophysical environment, which may alter the quantum yield and lifetime of the fluorophore(s). For example, epithelial tissues, generally have two primary sub-layers: a surface epithelium and an underlying stroma or submucosa; the specific fluorophores, as well as their concentration and distribution can vary significantly in these two tissue layers with a neoplastic change. Fluorescence spectroscopy of turbid media such as tissue also depends on the absorption and scattering that results from the concentration and distribution of nonfluorescent absorbers and scatterers, respectively, within the different sub-layers of the tissue.
The effect of the aforementioned factors on fluorescence spectroscopy of tissue is wavelength-dependent. First, the fluorophores that have absorption bands that lie in the same wavelength range as the excitation light will be excited and hence, emit fluorescence. The absorption and scattering properties of the tissue will affect light at both excitation and emission wavelengths. Therefore, only those fluorophores contained in the tissue layers to which the excitation light penetrates and from which, the emitted light can escape the tissue surface will produce measurable fluorescence. Elastic scattering events in tissue are caused by random spatial variations in the density, refractive index, and dielectric constants of extracellular, cellular and subcellular components. Tissue scattering generally decreases monotonically with increasing wavelength over the ultraviolet (UV), visible (VIS) and near-infrared (NIR) spectral regions.
Although absorption in tissue is strongly wavelength-dependent, it tends to generally decrease with increasing wavelengths. Consequently, the penetration depth of light, which is primarily a function of the tissue absorption properties, decreases from several centimeters to a few hundred microns, from the near infrared to the ultraviolet. For example, in the UV spectral region, the penetration depth of light in tissue is approximately 225 μm at 337 nm.
The illumination and collection geometry of the excitation and the emitted light, respectively, can also affect the fluorescence measurement from tissue, with respect to both the intensity and line shape. This may be attributed to the fact that although the fluorescence is generated approximately isotropically from the fluorophores within a medium, the fluorescence emitted from its surface may range from isotropic to anisotropic depending on whether the medium is highly absorbing, dilute or turbid. Monte Carlo simulations have been used extensively to simulate light distribution in turbid media to explore the effect of absorption and scattering on the fluorescence emitted from the surface, using finite excitation beam profiles and complex excitation and emission geometries.
Fluorescence measurements have been performed on biologic fluids, single cells, cell suspensions, frozen tissue sections and from bulk tissues, both in vitro and in vivo. The various types of instruments employed for these measurements, essentially have the same basic components. A schematic of the basic components of such an, instrument is shown in FIG. 3. It consists of a monochromatic excitation light source, a flexible, delivery and collection conduit for the delivery of excitation light to and the collection of the emitted light from the biologic medium, a dispersing element, which separates the emitted light into its respective wavelengths and a detector, which measures the intensity at the emission wavelength (s).
Generally, monochromatic excitation light sources are used and include ultraviolet and visible arc lamps (mercury, xenon) followed by a bandpass filter and continuous wave (Argon ion-ultra-violet lines, Helium—Cadmium-442 nm) or pulsed lasers (nitrogen-337 nm; the addition of dyes in an attached resonant cavity provides additional visible wavelengths). Lasers have the advantage of efficient coupling into fiber-optic probes. However, filtered arc lamps have the advantage of excitation wavelength tunability, when used with a series of band pass filters or a monochromator and they are generally, more portable. It should be noted that pulsed lasers, with very short pulse durations (in the order of nanoseconds) are necessary when the biologic medium needs to be illuminated with pulsed excitation light for gated detection (which provides effective rejection of ambient light during florescence measurements) and for fluorescence lifetime measurements.
Two approaches are used to illuminate and collect light from tissues. The first approach is to use fiber-optic probes that are placed directly in contact with the tissue (contact approach), and the second approach is to use a series of lenses to project the light onto the surface and collect it, in a similar manner (non-contact approach). With the contact approach, variable pressure on the biologic medium may distort the fluorescence spectrum. However, with the non-contact approach, the fluorescence intensity will vary with the variable, source-sample and sample-detector distance. In general, the contact approach is used for steady-state and time-resolved, fluorescence measurements from small tissue areas, whereas the non-contact approach is more suited for fluorescence imaging from relatively larger areas of tissue.
Light can be spectrally dispersed using a monochromator or a spectrograph, which are both dispersing components. A monochromator presents one wavelength or band pass at a time of the input light from its exit slit, whereas a spectrograph presents a range of wavelengths of the input light, simultaneously at the exit focal plane. Monochromators can be used as filters in conjunction with arc lamps to produce monochromatic excitation light at a series of wavelengths (if only several excitation wavelengths are needed, band pass filters are more appropriate). Monochromators can also be used to disperse the emitted light into its respective wavelengths, each of which can be detected serially using a single-channel detector. However, spectrographs can be used to disperse the emitted light into its respective wavelengths, simultaneously, for multi-channel detection.
The important considerations in choosing a detector are the type of measurements being made, i.e., single wavelength versus multi-wavelength and single-pixel (small area measurements) versus multi-pixel (large area measurements). Fluorescence measurements from single-pixels can be made either using a single-channel or multi-channel detector. If fluorescence intensity at only one or several wavelengths is being measured, single-channel, photo emissive tubes called photo multiplier tubes (PMT) or semi-conductor based, avalanche photodiodes (APD), with band pass filters can be used. For fluorescence spectroscopy, a spectrograph coupled to a multi-channel, photo diode array is appropriate. Fluorescence spectroscopy can also be performed using a monochromator coupled to a PMT. In the case of fluorescence imaging from multiple pixels, a two-dimensional, charged coupled device (CCD) camera, with band pass filters may be employed. To reduce or eliminate the detection of ambient light, a detector with an intensifier for fast gating (several nanoseconds) is used in conjunction with a pulsed excitation light source. Also, in order to minimize the detection of the back-scattered excitation light, which is much stronger than the weaker emitted light, optical components, such as long pass or dichroic filters may be employed in front of the detection system.
Single-pixel (<2 mm, diameter of tissue area) measurements of tissue fluorescence spectra, in vivo have been performed mostly using a pulsed excitation light source, a fiber-optic probe (contact approach), a spectrograph and an intensified photo diode array. The transient fluorescence decay profiles at a specific excitation-emission wavelength pair have also been measured using a similar instrument, except that the spectrograph and multi-channel, photo diode array have been replaced by a filtered, single-channel PMT or APD. Finally, fluorescence imaging from multiple pixels of tissue, in vivo (tissue area is a few centimeters in diameter) has been performed with a non-contact approach using a continuous wave laser in combination with a band pass filter and a CCD camera. Measurements with single-pixel and multi-pixel instruments generally require several seconds to a minute in a clinical setting.
Fluorescence spectroscopy in the ultraviolet and visible spectral regions has been developed and employed to differentiate diseased from non-diseased tissues, in vivo. The altered biochemical and morphologic state that occurs as tissue progresses from a non-diseased to diseased state, is reflected in the spectral characteristics of the measured fluorescence. This spectral information can be compared to tissue histology, the current gold standard, which indicates the absence or presence and grade of disease. Mathematical algorithms can then be developed and used to classify tissues into their respective histologic category, based on their spectral features. These mathematical algorithms can be implemented in software, thereby enabling fast, non-invasive, automated screening and diagnosis in a clinical setting.
There are generally two steps involved in the development of a mathematical algorithm, which is based on fluorescence spectroscopy. The first step is to dimensionally reduce the measured spectral variables. The second step is to develop a classification scheme for the discrimination of these useful spectral parameters into relevant histologic/histopathological categories. The development of current mathematical algorithms based on fluorescence spectroscopy can be classified broadly into three categories: 1) algorithms based on qualitatively selected spectral variables (fluorescence intensities at several emission wavelengths), 2) algorithms based on statistically selected spectral parameters (a more robust evaluation and use of all the measured spectral information) and 3) algorithms based on parameters that reflect the biochemical and/or morphologic features of the tissue. Classification schemes employ either a binary or probability based discrimination. In most cases, algorithms are based on qualitatively or statistically selected spectral variables in conjunction with binary classification methods.
While current fluorescence spectroscopy and imaging methods detect neoplastic tissue areas, their sensitivity to the neoplastic layer in tissue is limited by how much of the probing volume intercepts the target of interest. In other words, the probing depth is fixed by the illumination and collection geometry. However, the depth and thickness of the neoplastic growth can vary and the sensing volume may not be optimized for maximal contrast. Being able to maximize the contrast between the neoplastic growth and normal tissue will significantly enhance the diagnostic capability of this technology.